The hypoxia (HYPX)-mediated excessive generation of mitochondrial free reactive oxygen species (mROS) and the overload Ca2+ influx via the inhibition of TRPV4 are controlled by the treatment of antioxidants. However, the molecular mechanisms underlying melatonin (MLT)'s neuroprotection remains elusive. We investigated the role of MLT via modulation of TRPV4 on oxidative neurodegeneration and death in SH-SY5Y neuronal cells. The SH-SY5Y cells were divided into five groups as follows: control, MLT (1 mM for 2 h), HYPX (200 mu M CoCl2 for 24 h), HYPX + MLT, and HYPX + TRPV4 blockers (ruthenium red-1 mu M for 30 min). The HYPX caused to the increase of TRPV4 current density and overload Ca2+ influx with an increase of mitochondrial membrane potential and mROS generation. The changes were not observed in the absence of TRPV4. When HYPX exposure and TRPV4 agonist (GSK1016790A)-induced TRPV4 activity were inhibited by the treatment of ruthenium red or MLT, the increase of mROS, lipid peroxidation, apoptosis, Zn2+ concentrations, TRPV4, caspase-3, caspase-9, Bax, and Bcl-2 expressions were restored via upregulation of reduced glutathione, glutathione peroxidase, and total antioxidant status. The levels of apoptosis and cell death in the cells were enriched with increases of caspase-3 and-9 activations, although they were decreased by MLT treatment. In conclusion, the treatment of MLT modulates HYPX-mediated mROS, apoptosis, and TRPV4-mediated overload Ca2+ influx and may provide an avenue for protecting HYPX-mediated neurological diseases associated with the increase of mROS, Ca2+, and Zn2+ concentration.